Field of the Invention
The present invention relates to methods, devices, and systems for fluid processing in a microfluidic device. More particularly, aspects of the present invention relate to methods, devices, and systems utilizing a single slug approach in which a single slug fills a thermal zone of a microfluidic channel, and the thermal zone is used for both PCR amplification melt data acquisition.
Description of the Background
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, identification of crime scene features, the ability to propagate industrial organisms, and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer.
One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying deoxyribonucleic acid (DNA). With PCR, one can produce millions of copies of DNA starting from a single template DNA molecule. The PCR process includes phases of “denaturation,” “annealing,” and “extension.” These phases are part of a cycle which is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
The PCR process phases of denaturing, annealing, and extension occur at different temperatures and cause target DNA molecule samples to replicate themselves. Temperature cycling (thermocyling) requirements vary with particular nucleic acid samples and assays. In the denaturing phase, a double stranded DNA (dsDNA) is thermally separated into single stranded DNA (ssDNA). During the annealing phase, primers are attached to the single stranded DNA molecules. Single stranded DNA molecules grow to double stranded DNA again in the extension phase through specific bindings between nucleotides in the PCR solution and the single stranded DNA. Typical temperatures are 95° C. for denaturing, 55° C. for annealing, and 72° C. for extension. The temperature is held at each phase for a certain amount of time, which may be a fraction of a second up to a few tens of seconds. The DNA is doubled at each cycle, and certain applications may require numerous (e.g., 20 to 40 or more) cycles to produce enough DNA. To have good yield of target product, one has to accurately control the sample temperatures at the different phases to a specified degree.
The multi-slug approach is a high throughput approach to performing PCR and other amplification reactions in microfluidic devices, as well detecting and analyzing amplified nucleic acids in or on the devices. The multi-slug approach creates a sequence of short slugs (i.e., a slug train) in a microfluidic channel of a microfluidic device. Several slugs share a PCR thermal zone (“Zone 1”) in which the temperature of the slugs is cycled to perform a PCR amplification before passing into a melt thermal zone (“Zone 2”) in which the temperature of the slugs is ramped to melt any DNA in the slugs (i.e., dissociate the strands of any DNA in the slugs).
A multi-slug system may have a cartridge with one or more microfluidic channels. Each of these channels may have a three branched (i.e., Y-shaped or T-shaped) topology that is used to created short slugs inside the PCR chip. The slugs alternate between a test/sample slug and a flow marker or “blanking” slug. Fluorescence emitted from the blanking slugs may have a different color than fluorescence emitted by the sample slugs. The color difference enables the blanking slugs to be spatially imaged and used for slug position sensing. Information about the slug position may be fed back to adjust the pressure applied to the downstream end of the channel and, thereby, control the positions of the slugs.
FIG. 11 illustrates an exemplary timing sequence of slugs progressing through a channel of a reaction chip (“Uchip”) of a system implementing a simplified multi-slug approach in which no more than two sample slugs are in the PCR thermal zone (Zone 1) at a given time. TABLE 1 (below) shows the events occurring during steps of a simplified multi-slug processing in a multi-slug system having a microfluidic device with an interface chip and a reaction chip. In Table 1 below, important events are shown in bold.
TABLE 1InterfaceReactionChip FlowChip FlowZone 1Zone 2ExemplaryStep #EventsEventsEventsEventsTime (s)1—Pull InPCR—100Sample Slug2Load BlankingHold SamplePCR—100SolutionSlug3—Pull InPCR—100BlankingSlug4Load SampleHoldPCRMelt100SolutionBlankingSlug
As illustrated in FIG. 11 and in TABLE 1, the multi-slug system is highly coupled, with multiple important events occurring at the same time. As multiple important events occur at the same time, there is a need to balance flow, PCR, and melt requirements, and no single event is truly optimized.
The design concept of the multi-slug approach was based on having multiple slugs in the PCR thermal zone (Zone 1) to increase throughput. However, in practice the multi-slug approach may have some drawbacks. The multi-slug timing may be problematic because of the need to always compromise and balance the conflicting requirements of two or three concurrent events. The multi-slug approach also uses at least two sets of slugs (i.e., two samples and two blanks) on the reaction chip at the same time. Furthermore, the small slugs may be difficult to align, which may lead to the need for a time consuming slug training step (e.g., using start-up slugs).
The following describes some shortcomings that may be present in the multi-slug design:
1. Several slugs in a channel share the PCR thermal zone (Zone 1) at the same time, which means the same thermocycling conditions must be used for all of the slugs in the PCR thermal zone. This may be limiting to assay optimization.
2. As the PCR thermal zone (Zone 1) is cycling the temperatures to perform PCR amplification as the slugs pass through the entirety of the PCR zone thermal, the temperature in the PCR thermal zone must be uniform across the entirety of PCR thermal zone, which may be, for instance, 14.75 mm in length. However, obtaining a uniform temperature across the entirety of PCR thermal zone may be difficult due to thermal gradients, variations in heat sink attachment, etc.
3. Slugs spend a portion of the thermal cycling time straddling the leading and trailing edges of PCR thermal zone (Zone 1). See, e.g., FIG. 11. This results in a varying number of cycles for different portions of the slug. Moreover, temperature may be less uniform at the edges of the PCR thermal zone, and portions of the slug are subjected to thermal cycling at non-optimal temperatures which may result in non-specific amplification and/or overall low efficiency.
4. The design is not compatible with hot starting the polymerase enzyme or providing a longer initial denaturation step because several slugs share the heater of the PCR thermal zone (Zone 1) at the same time.
5. Simultaneously running PCR thermal heaters in the PCR thermal zone (Zone 1) and melt thermal heaters in the melt thermal zone (Zone 2) presents certain challenges. For example, a pumping effect is caused by the expansion of an aqueous solution in a channel when heated. Rapid cycling of the PCR thermal zone (Zone 1) temperature produces an oscillatory pumping effect on the fluid in the channel that, in some circumstances, can adversely affect the quality of melt data obtained from the melt thermal zone (Zone 2).
6. Slug length varies as slugs cross the chip due to variations in the depth of the microfluidic channels. In other words, slugs lengthen or shorten if the microchannels become more or less shallow. The variation in slug length makes accurate positioning of the slugs in the melt thermal zone (Zone 2) problematic, and a complex and time consuming position calibration approach may be required to mitigate the variation in slug length.
7. The short slugs used in the multi-slug approach may have a greater propensity for unwanted diffusion effects (e.g., amplicon carryover).
8. PCR product that is created in the PCR thermal zone (Zone 1) may not make it to the melt thermal zone (Zone 2) for analysis because of dispersion that occurs as the fluid is moved.
9. The multi-slug system requires continuous active feedback control of the pressure applied to the downstream end of the reaction chip. If for any reason control of the pressure fails, the slug train in a channel can undergo a loss of positioning control. Generally speaking, there may be no way to recover use of the particular channel for the slug train for which pressure control was lost.
10. Any motion that may exist due to active continuous flow control may result in significant artifacts in melt curves as the short slug length means the product and fluorescence change rapidly.
11. In certain color imaging systems, there may be cross-talk between the color planes. Having short slugs of different color may increase the cross-talk and bleed over between colors.
12. Changing the flow rates requires a corresponding change in PCR and melt timing. This may result in un-wanted delays where the fluid sits before the melt (perhaps in a region of the chip which is not rigorously controlled in terms of temperature).
13. Changing the PCR timing requires a corresponding change in the flow rates. This is undesirable because the microfluidics may not function as reliable at the flow rates necessitated by the PCR timing.
14. There may be no time for a long melt or repeating a melt because the slug train must keep advancing.
15. The multi-slug system may be slow to start up because of the difficulty in creating the first short slugs and the need for slug position calibration.
16. The multi-slug system may be slow to reflex to a melt result because slugs are already in the queue.
17. Two thermal zones (i.e., a PCR thermal zone and a different melt thermal zone) may require twice as much hardware: data acquisition, signal conditioning, heat sink, cabling, etc.
18. The small size of the slugs, dispersion, and motion across PCR thermal zone (Zone 1) may make real time PCR highly problematic.
Accordingly, there is a need for improved methods, devices, and systems for processing slugs in microfluidic devices.